Pharmacological composition for preventing neurotoxic side effects of NMDA antagonists

ABSTRACT

This invention discloses mixtures of NMDA antagonists and anti-cholinergic agents, which can be used to prevent excitotoxic damage in the central nervous system or for anesthetic purposes in human or veterinary medicine. Anti-cholinergic agents such as scopolamine, atropine, benztropine, trihexyphenidyl, biperiden, procyclidine, benactyzine, or diphenhydramine can be used in conjunction with, or subsequent to, administration of an NMDA antagonist such as MK-801. The NMDA antagonist exerts a primary protective effect by preventing or reducing excitotoxic damage due to stroke, perinatal asphyxia, and various other types of injury or disease; however, strong NMDA antagonists such as MK-801 can also cause neurotoxic side effects, including vacuole formation, mitochondrial dissolution, and neuronal death in certain types of neurons such as cingulate/retrosplenial cerebrocortical neurons. The anti-cholinergic agent will reduce or eliminate those damaging side effects, without interfering with the primary protective value of the NMDA antagonist. The anti-cholinergic agents described herein can also reduce the toxic side effects associated with illegal use of drugs such as phencyclidine (also known as PCP or angel dust).

This application is a divisional application of U.S. application Ser.No. 424,548, filed on Oct. 20, 1989, now Pat. No. 5,034,400.

BACKGROUND OF THE INVENTION

This invention is in the fields of pharmacology and neurology. Itrelates to compounds and methods for protecting the central nervoussystem against neurotoxic side effects of certain therapeutic drugs andagainst neurodegenerative disease processes.

Receptors, messenger molecules, agonists, and antagonists

The surfaces of nerve cells in the central nervous system (the CNS,which includes the brain, spinal cord, and retina) contain various typesof receptor molecules. In general, a receptor molecule is a polypeptidewhich straddles a cell membrane. When a messenger molecule interactswith the exposed extracellular portion of the membrane receptormolecule, it triggers a difference in the electrochemical status of theintracellular portion of the receptor, which in turn provokes someresponse by the cell. The messenger molecule does not bond to thereceptor; instead, it usually disengages from the receptor after a briefperiod and returns to the extracellular fluid. Most receptor moleculesare named according to the messenger molecules which bind to them.

An "agonist" is any molecule, including the naturally occurringmessenger molecule, which can temporarily bind to and activate a certaintype of receptor. An agonist can cause the same effect as the naturalmessenger molecule, or in some cases it can cause a more intense effect(for example, if it has a tighter affinity for the receptor molecule andremains bound to the receptor for a prolonged period).

By contrast, an "antagonist" is a molecule which can block or reduce theeffects exerted by the natural messenger molecule. This can happen inseveral different ways. A "competitive antagonist" binds to a certaintype of receptor without triggering it, thereby preventing the naturalmessenger molecule from reaching and activating the receptor. A"non-competitive antagonist" functions in other ways. For example, areceptor referred to as the PCP receptor, which is triggered bymolecules such as PCP or MK-801, apparently can override the effects ofa different type of receptor, the NMDA receptor (both receptors arediscussed below). Therefore, PCP and MK-801 are regarded asnon-competitive antagonists for the NMDA receptor.

The role a certain molecule plays as an agonist or antagonist must beviewed with regard to a certain type of receptor. For example, whileMK-801 is an antagonist for the NMDA receptor, it is an agonist for thePCP receptor. Most agonists and antagonists are xenobiotic drugs, i.e.,they do not exist naturally in the body. For more information onneuroanatomy, neurotransmitters, receptors molecules, and agonists andantagonists which interact with CNS receptors, see Adelman 1987(complete citations are provided below).

The two main classes of excitatory receptor molecules are referred to as"cholinergic" receptors and "glutamate" receptors. Both types ofreceptors are present in the synaptic junctions that serve as pathwaysfor impulses between CNS nerve cells. Most other types of receptors inthe CNS involve inhibitory neurotransmitters.

Excitatory amino acids and neurotoxicity

Accumulating evidence implicates excitatory amino acids (EAA's) such asglutamate and aspartate as causative agents in certain types of CNSdamage associated with epsilepsy, hypoglycemia, hypoxia/ischemia(stroke, cardiac arrest, perinatal asphyxia), alcoholism, and trauma ofthe brain or spinal cord. It is also believed that EAA's may be involvedin slowly developing neurodegenerative disorders such as Huntington's,Parkinson's and Alzheimer's diseases. Glutamate and aspartate (the ionsor salts of glutamic acid and aspartic acid) are found naturally in highconcentrations in the central nervous system (CNS) where they functionas excitatory neurotransmitters.

Although these substances are beneficial and of critical importance forthe normal functioning of the CNS, under abnormal conditions they candestroy CNS neurons by an "excitotoxic" process. Excitotoxicity refersto the process whereby EAA's that are released from one neuronexcessively stimulate (excite) receptor molecules located on theexternal surface of another neuron. Excitotoxicity also refers to thesame excitatory neurotoxic process when triggered by glutamate or EAAanalogs of glutamate ingested in foods or administered systemically tovarious mammalian species. Glutamate and asparate are sometimes called"endogenous" excitotoxins, meaning that they are excitatory neurotoxinscontained naturally within the CNS, whereas EAA ingested in foods oradministered systemically are referred to as "exogenous" excitotoxins.For an extensive review, see Olney 1989.

EAA receptors, also known as glutamate receptors, are categorized intothree subtypes, each named after a glutamate analog which selectivelyexcites them: N-methyl-D-aspartate (NMDA), kainic acid (KA), andquisqualate (QUIS). Glutamate is capable of activating all threereceptor subtypes.

Normally, relatively high glutamate concentrations (in the general rangeof about 10 mM) are maintained inside cells of the CNS, but highconcentrations are not allowed in the extracellular fluid whereglutamate can exert excitotoxic action at EAA receptors. After glutamateis released by a neuron for neurotransmitter purposes, it normally istransported back inside a cell by means of a transport mechanism whichrequires energy. Under severe low energy conditions such ashypoglycemia, hypoxia, or ischemia, the transport systems may lacksufficient energy to transfer extracellular glutamate back into thecell, so the glutamate accumulates at abnormal levels and excessivelystimulates the EAA receptors. This can lead to continuous neuronaldischarge, which in turn causes additional glutamate release andextracellular accumulation of excess glutamate, leading to a cascade ofincreasing neurotoxic injury, which can result in death or permanentdamage to the brain.

Other mechanisms by which EAA's can cause neuronal injury includeabnormal sensitivity of EAA receptors to the excitatory action of EAA's,and the presence of abnormal molecules (such as glutamate analogs,certain types of food poisons, etc.) with excitotoxic properties. Suchreceptor-triggering molecules can accumulate at EAA receptors becausethey are not recognized by the cellular transport systems as moleculeswhich should be removed from the extracellular fluid.

In these neurotoxic situations, one method of preventing or minimizingexcitotoxic injury to the neurons involves administering drugs thatselectively block or antagonize the action of the excitotoxic moleculesat the EAA receptors.

NMDA antagonists as neuroprotective drugs

The EAA receptor subtype that has been implicated most frequently inneurodegenerative diseases and neurotoxicity is the NMDA receptor. Anentire issue of Trends in Neurosciences (Vol. 10, Issue 7, July 1987)was devoted to review articles pertaining to the NMDA receptor, and toNMDA "antagonists" (i.e., molecules which can block or reduce theeffects of NMDA at NMDA receptors). Agents which act by binding directlyto NMDA receptors, such as D-2-amino-5-phosphonopropanoate (D-AP5) andD-2-amino-7-phosphonoheptanoate (D-AP7), are referred to as competitiveNMDA antagonists. Those two compounds are of limited therapeutic utilitybecause they do not readily penetrate the blood-brain barrier. However,it is possible that some recently developed competitive NMDAantagonists, such as the Ciba-Geigy compound CGS 19755 (Boast, 1988) or3-(2)-carboxypiperazin-4-yl)-propyl-1-phosphonate (CPP) or itsunsaturated analog, CPP-ene, may affect the CNS following systemicadministration (Herrling et al 1989).

The most powerful and effective NMDA antagonists known at the presenttime act at another receptor, the phencyclidine (PCP) receptor, which isconsidered a component of an ion channel complex that involves the NMDAreceptor (Kemp et al 1987). These compounds are called non-competitiveNMDA antagonists because they do not compete for binding sites at NMDAreceptors. When phencyclidine or its analogs activate the PCP receptor,the flow of ions through the NMDA ion channel is blocked orsubstantially reduced, so that when the NMDA receptor is activated by anEAA, the NMDA receptor response does not result in the flow of ioncurrents. This blocks the excitation of the neuron.

Four compounds which can activate the PCP receptor, and which thereforeserve as non-competitive NMDA antagonists, are phencyclidine, MK-801,ketamine, and tiletamine. Each is discussed in more detail below. Allfour of these agents can penetrate the blood-brain barrier.

MK-801, a phencyclidine analog manufactured by Merck, Sharp and Dohme(Rahway, N.J.) is believed to be the most powerful PCP agonist of thefour compounds listed above (Olney et al 1987). It has generated greatinterest recently, largely due to its potential for reducing neurotoxicdamage involving NMDA receptors. The chemical name for MK-801 is(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-iminemaleate.

Neurological problems that might be aided by NMDA antagonists

Non-competitive NMDA antagonists have been shown in in vivo animalexperiments (reviewed in Olney 1989) to protect CNS neurons againstdamage caused by persistent seizures, hypoglycemia, hypoxia/ischemia,trauma, thiamine deficiency, and methamphetamine poisoning (a form ofneurotoxicity related to Parkinson's disease). It is possible,therefore, that such agents might be used therapeutically forneuroprotective purposes in conditions such as the above.

It is also possible that NMDA antagonists might be used to prevent braindamage associated with alcoholism. In chronic alcoholics, neuronaldegeneration has been described in several regions of the brain,including periventricular and periaqueductal regions, the thalamus, thehypothalamus, and mammillary bodies. Individuals with this type of braindamage manifest a form of dementia known as Wernicke/Korsakoff syndrome,which includes severe deficits in memory and cognitive functions. It isbelieved that this syndrome relates to dietary deficiencies, especiallythiamine (vitamin B) deficiency. It is known that people who suffer fromthiamine deficiency are subject to the same pattern of brain damage andthe same dementing Wernicke/Korsakoff syndrome that is seen inalcoholism. Recently, it was demonstrated that in a rat model ofthiamine deficiency, which entails disseminated brain damage distributedin a Wernicke/Korsakoff pattern, the brain damage can be markedlyattenuated by pretreatment with MK-801 (Langlais et al 1988). Thatresult suggests that this type of brain damage is mediated by anexcitotoxic mechanism involving the NMDA receptor ion channel complex,and that patients with acute symptoms suggesting an impendingWernicke/Korsakoff syndrome might benefit from treatment with an NMDAantagonist such as MK-801.

It is also suspected that there may be a link between virally-inducedneurodegenerative conditions and NMDA receptor-mediated excitotoxicity(Olney 1989). When a viral infection triggers changes in neuralhomeostasis, endogenous excitotoxins such as glutamate and aspartate maybecome involved in cell death or damage. Therefore, NMDA antagonistssuch as MK-801 may be useful for preventing neuronal degenerationassociated with viral infections that involve the CNS, such asJacob-Creutzfeldt syndrome and encephalitis associated with herpes ormeasles infection.

NMDA antagonists such as MK-801 might also be useful for protectingagainst brain damage associated with a newly recognized type of foodpoisoning. In January 1988, there was an outbreak of food poisoning inCanada affecting 145 identified individuals, some of whom died and werefound at autopsy to have widespread brain damage (Quilliam et al 1989).Some of the surviving victims suffered severe brain damage and becamepermanently demented. All of the victims had ingested mussels from theNewfoundland region. Analysis of the mussels revealed a highconcentration of domoate, a powerful convulsant which apparently causesbrain damage by inducing persistent seizure activity which releasesexcessive glutamate, triggering an excitotoxic cascade. It is believedthat domoate poisoning may become a recurrent problem in several regionsof the world. The inventor of the subject invention has recentlydemonstrated that certain NMDA antagonists (including MK-801 andphencyclidine) protect against domoate neurotoxicity. Therefore, NMDAantagonists such as MK-801 may serve as antidotes to prevent braindamage, dementia and/or death in domoate poisoning.

In addition to seizure-related brain damage associated with domoatepoisoning, brain damage also occurs as a result of persistent seizuresin patients with epilepsy. In this situation also, it is believed thatexcessive activation of NMDA receptors by endogenous glutamate may causeor exacerbate brain damage. It is possible, therefore, that patients whocome to emergency rooms in status epilepticus (a state of continuousepileptic seizure activity) might be protected from permanent braindamage by timely treatment with an NMDA antagonist.

However, the potential therapeutic uses of MK-801 and other NMDAantagonists must be viewed with caution, because it has recently beendiscovered (Olney et al 1989) that such agents can inflict their owntype of neurological damage.

The subject invention, as explained below, involves a class ofprotective agents that can be administered along with NMDA antagonistssuch as MK-801, to reduce or eliminate the dangers and deleterious sideeffects of NMDA antagonists. The subject invention thereby enables thesafe use of NMDA antagonists such as MK-801 to accomplish the beneficialresults set forth above.

Neurotoxic side effects of NMDA antagonists

A potentially serious side effect of MK-801, phencyclidine, and relateddrugs is that they may induce a neurodegenerative reaction in theposterior cingulate and retrosplenial cerebral cortex, even whenadministered in relatively low doses (Olney et al 1989). In a series ofexperiments, MK-801 and phencyclidine were given to adult rats to testfor neuroprotection against seizure-related brain damage. Those agentsdid protect neurons in certain brain regions from seizure-relateddamage, but they also caused a different type of neurotoxic reaction inother brain regions, the posterior cingulate and retrosplenial cerebralcortices. The neurotoxic reaction, which was observed during microscopicanalysis of CNS tissue after the rats were sacrificed, consisted of theformation of vacuoles (membrane-enclosed spaces in the cytoplasm thatare not present in normal cells) and the dissolution of mitochondria(energy-producing organelles inside the cells). Although these changesappeared to be reversible if the doses of MK-801 or phencyclidine weresufficiently low, it has recently been discovered that irreversiblenecrosis of cingulate cortical neurons follows the administration of 5mg/kg MK-801.

In adult rats, the ED₅₀ for producing vacuoles in cingulate neurons byMK-801 administration (i.e., the dosage of MK-801 which will producevacuoles in 50% of the animals treated) is 0.18 mg/kg, administeredintraperitoneally (ip; Olney et al 1989). Since the doses of MK-801 usedin animal experiments for protecting neurons against ischemic braindamage usually are in the range of 1 to 10 mg/kg, it appears that theuse of MK-801 for therapeutic neuroprotection poses a major risk ofinducing potentially serious neurotoxic side effects.

The mechanism by which MK-801, phencyclidine and related drugs causevacuole formation in cingulate/retrosplenial neurons is poorlyunderstood. However, recent evidence that this effect can be reproducedby microinjection of a competitive NMDA antagonist (D-AP5) into thecingulate cortical region (Labruyere et al 1989) suggests that any agentthat antagonizes the NMDA receptor ion channel complex by any mechanismmay have this toxic property, since the vacuole reaction occurs whenNMDA receptor function is suppressed, either by direct antagonistbinding of D-AP5 to the NMDA receptor, or by interaction of MK-801 atthe level of the NMDA receptor ion channel. An important implication ofthis finding is that some recently developed competitive NMDAantagonists which may be able to penetrate the brain in sufficientconcentration to be used as neuroprotective drugs, such as CGS 19755,CPP, and CPP-ene, may not provide an acceptable alternative to thenon-competitive NMDA antagonists, since both groups of compounds mightcause the same type of neurotoxic side effect. These findings andimplications suggest that both groups of compounds would be moreacceptable for therapeutic purposes if a method were found that preventstheir neurotoxic side effects without interfering with theirneuroprotective actions.

History of the uses and abuses of NMDA antagonists

Phencyclidine (PCP) was originally introduced into clinical medicinesome 30 years ago as an anesthetic (Goodman and Gilman 1975). Shortlythereafter, it was withdrawn from the market because it was found tohave hallucinogenic properties which invited illicit use by drugabusers. Since then, PCP (also known as angel dust) has becomeincreasingly popular as a "recreational" drug and currently is a majorcause of drug-induced psychotic reactions (which occasionally lead toextremely violent crimes) among drug abusers. The pathomorphologicaleffect of PCP (vacuole formation and mitochondrial dissolution incertain types of neurons) might be related to the mechanism by which PCPcauses toxic psychoses. Thus, if a drug could be found that prevents thepathomorphological effects of PCP, it might also prevent or amelioratethe psychotomimetic effects of PCP. Such a drug might be used inemergency rooms, or perhaps by the police, as an antidote to reduce boththe neurological damage and the psychotic effects of PCP in drugabusers.

Ketamine, a drug manufactured by Parke Davis and marketed under thetrade name Ketalar, is currently used both in human and in veterinarymedicine as an anesthetic. Ketamine is known to activate PCP receptorsand, like PCP and MK-801, is recognized as a non-competitive antagonistof the NMDA receptor-ion channel complex (Kemp et al 1987). Ketamine wasamong the drugs recently shown to produce pathomorphological effects oncingulate/retrosplenial neurons following intraperitoneal (ip)administration to rats (Olney et al 1989). Ketamine is known to inducean acute transient psychosis (called an "emergence" reaction) in about13% of human patients anesthetized with this agent (Physicians DeskReference, 1986). It has been proposed that the psychotic effects ofketamine, like those of PCP, may be psychological manifestations of thesame toxic process that causes pathomorphological changes incingulate/retrosplenial neurons, in which case a drug that could preventthe pathomorphological changes might also prevent or reduce thepsychotic manifestations. Even without preventing the psychoticmanifestations, eliminating the risk of pathomorphological changes wouldbe a significant benefit.

Tiletamine is a drug manufactured by A. H. Robins. It is currently usedin veterinary medicine, and is widely used for anesthesia on house pets.Tiletamine, like PCP, MK-801 and ketamine, is known to activate PCPreceptors and is recognized as a non-competitive antagonist of the NMDAreceptor-ion channel complex. Tiletamine was among the drugs recentlyshown to produce pathomorphological effects on cingulate/retrosplenialcortical neurons following ip administration to rats (Olney et al 1989).

MK-801, a drug manufactured by Merck, Sharp and Dohme and referred to asdizocilpine, was initially proposed as an anticonvulsant, but afterbrief human clinical trials, it was withdrawn from further testingseveral years ago with no published explanation. At about the same time,it was discovered that MK-801 is a potent activator of PCP receptors(with higher affinity for the PCP binding site than PCP itself; MK-801is more specific for PCP receptors than any other known compound), andthat PCP receptors are an integral component of the NMDA receptor ionchannel complex (Kemp et al 1987). It was also found that both MK-801and PCP block the excitatory effects of NMDA on neurons in the in vivorat spinal cord (Lodge et al 1987). In neurotoxicology studies, using anex vivo chick embryo retina assay, it was shown that MK-801 isapproximately 5-10 times more powerful than PCP in preventing theneurotoxic effects of NMDA on retinal neurons (Olney et al 1987).Previously, PCP had been recognized as the most powerful knownantagonist of NMDA neurotoxicity (Olney et al 1986).

Because of its great potency and the ease with which it penetrates bloodbrain barriers, MK-801 has become the drug used most widely in animalexperiments aimed at testing the neuroprotective properties of NMDAantagonists. Since it has now been shown to protect CNS neurons againstvarious degenerative processes that are thought to involve excessiveactivation of NMDA receptors (e.g., hypoxia/ischemia, prolongedseizures, hypoglycemia, thiamine deficiency, head or spinal cord trauma)there is considerable interest in using MK-801 for neuroprotectivepurposes in clinical neurology. Clearly, it would be desirable to have ameans of preventing the toxic action of MK-801 oncingulate/retrosplenial cortical neurons, thereby making this drugavailable for human therapy with reduced risk of neurotoxic sideeffects.

Cholinergic receptors

Cholinergic receptors are activated by acetylcholine, a relatively smallmolecule released by certain types of brain cells. Cholinergic receptorsare divided into two main classes: muscarinic and nicotinic.

Little is known about nicotinic receptors in the CNS. They exist in theperipheral nervous system, at neuromuscular junctions, and they arepresumed to exist inside the brain, but very limited progress has beenmade in developing agonist or antagonist molecules that can penetrateblood-brain barriers and be used to pharmacologically characterizenicotinic receptors that may exist in the brain.

Muscarinic receptors are subdivided into M1 and M2 receptors, based onthe discovery that pirenzepine binds with much greater affinity to onesubpopulation (M1) found primarily in the forebrain, than to a separatesubpopulation (M2) that exists primarily in the hindbrain and in theperipheral nervous system. Most anti-cholinergic molecules, althoughmore powerful than pirenzepine in binding to cholinergic receptors, donot show as high a degree of specificity for one receptor subpopulation.Thus, the anti-cholinergic agents of primary interest herein havesubstantial affinity for both M1 and M2 receptors (Burke 1986; Freedmanet al 1988). It is not known whether or to what extent theseanti-cholinergics also interact with nicotinic receptors inside thebrain, since reliable methods for identifying and characterizingnicotinic receptors in the brain have not been available.

Pilocarpine, a cholinergic agonist used in epilepsy research, has beenshown to cause seizures and seizure-related brain damage (Turski et al1983; Clifford et al 1987). Although the inventor has used MK-801successfully to prevent brain damage associated with seizures induced byvarious methods, a set of experiments described in Example 5 indicatesthat MK-801 has a potentiating effect when administered along withpilocarpine. The term "potentiate" refers to the fact that the MK-801lowered the seizure threshold and made test animals susceptible toseizures at a pilocarpine dosage that would not have caused seizures inthe absence of the MK-801. This finding raises questions about whetherNMDA antagonists would tend to induce seizures in humans who suffer fromepilepsy.

The inventor also discovered that pilocarpine and MK-801, whenadministered together, increase the formation of vacuoles incingulate/retrosplenial neurons. If an adult rat is treated withpilocarpine (75 mg/kg ip), the ED₅₀ of MK-801 for producingcingulate/retrosplenial vacuoles is reduced from 0.18 mg/kg to 0.05mg/kg.

Judging from these results, MK-801 apparently can exert one type ofbeneficial anti-convulsant effect, by blocking one of the majorexcitatory transmitter systems, the NMDA receptor system. However,MK-801 appears to potentiate another type of seizure activity mediatedby the cholinergic receptor system. These results imply that some kindof mechanism exists by which the cholinergic and NMDA receptor systemsare linked, such that drugs affecting either system can influenceneurological disorders such as seizure activity and formation ofvacuoles in cingulate/retrosplenial neurons.

Anti-cholinergic agents

A group of agents classified as anti-cholinergics (i.e., they block theactivation of cholinergic receptors) have been used in clinicalneurology as anti-parkinsonian drugs (Goodman and Gilman 1975). Theseagents were recently found by the inventor to protect rats against theconvulsant and brain damaging action of pilocarpine and anothercholinergic neurotoxin, soman (Price et al 1989). The drugs thatconferred this neuroprotective action are procyclidine, biperiden andtrihexyphenidyl, which are structurally related compounds of thearyl-cyclo-alkanolamine class. These agents, especially biperiden andtrihexyphenidyl, are considered cholinergic antagonists that act quitepowerfully at the M1 muscarinic receptor (Freedman et al 1988), whichsuggests a possible explanation for their efficacy in blocking theneurotoxic actions of pilocarpine, which is primarily an M1 cholinergicagonist. These aryl-cycloalkanolamines have also been shown to havelimited effectiveness as NMDA receptor antagonists (Olney et al 1987),but they are considered much more powerful as M1 cholinergic antagoniststhan as NMDA antagonists.

Procyclidine, which has some degree of affinity for NMDA receptors inaddition to being an anti-cholinergic agent, can be administered toadult rats at a high dose (75 mg/kg) without producing neurotoxic sideeffects such as cingulate/retrosplenial vacuole formation induced byother NMDA antagonists such as MK-801 or D-AP5. Procyclidine isdescribed in U.S. Pat. No. 2,891,890 (Adamson 1959), and is marketedunder the trade name "Kemadrin" by Burroughs-Wellcome.

Biperiden has been studied for its mood altering effects (Fleischhackeret al 1987) and for its interaction with muscarinic receptors (Syvalahtiet al 1987). The hydrochloride salt of biperiden has been studied forits interaction with nicotine and oxotremorine in rat diaphragm (Das etal 1977). Biperiden is marketed under the trade name "Akineton" byKnoll. Triperiden is marketed in Europe under the trade name "Norakin"by VEB Fahlberg-List (Magdeburg, West Germany).

Trihexyphenidyl has been studied for its effects in schizophrenicpatients (Hitri et al 1987) and for its effects on memory in elderlypatients (McEvoy et al 1987). It is marketed under the trade name"Artane" by Lederle, and is used to reduce Parkinson symptoms inschizophrenics who are being treated with phenothiazine compounds.

Various other aryl-cycloalkyl-alkanolamine compounds have also beenstudied for varying purposes (e.g., U.S. Pat. Nos. 4,031,245 and3,553,225, West German Offen. No. 1,951,614, and Mann et al 1976).However, none of the research with this class of compounds involvestheir use for reducing the neurotoxic effects of PCP, MK-801, or otherNMDA antagonists.

A number of other compounds are known to function as anti-cholinergicagents. Benztropine, sold under the trade name "Cogentin" by Merck,Sharp and Dohme, is used to reduce Parkinson symptoms in schizophrenicsbeing treated with phenothiazine compounds (Vernier 1981). Benactyzineis used in conjunction with meprobamate in a formulation called"Deprol," sold by Wallace Laboratories (Physicians Desk Reference 1989,p. 2200). Scopolamine and atropine, both of which have been used widelyin medicine for anesthesia-related purposes, have also been used asanti-Parkinsonian drugs, but they tend to cause side effects whenadministered at anti-Parkinson dosages, due to their interactions withcholinergic receptors in the peripheral nervous system. In summary,accumulating evidence suggests that NMDA antagonists might be highlyuseful therapeutic agents in various neurological disorders. However,prior to this invention, there was no known agent or method forpreventing certain deleterious side effects of those NMDA antagonists.

One object of this invention is to provide a pharmacological agent andmethod which can be used in human medicine to reduce the neurotoxicityof NMDA antagonists.

Another object of this invention is to provide an agent and a method forreducing the neurotoxicity of agents such as ketamine and tiletamine,which are used as veterinary anesthetics on animals including housepets.

Another object of this invention is to provide a mixture of an NMDAantagonist combined with a protective agent which reduces the neurotoxiceffects of the NMDA antagonist. Such mixtures can be used to prevent orminimize deleterious CNS effects associated with various neurologicaldisease processes.

SUMMARY OF THE INVENTION

This invention involves drugs that can be used to reduce the neurotoxicside effects that can be caused when NMDA antagonists are used asanti-convulsants, to prevent excitotoxic damage in the central nervoussystem, or for anesthetic purposes in human or veterinary medicine. Thismethod involves the use of anti-cholinergic agents such as scopolamine,atropine, benztropine, trihexyphenidyl, biperiden, procyclidine,benactyzine, or diphenhydramine in conjunction with, or subsequent to,administration of an NMDA antagonist such as MK-801. Theanti-cholinergic agents greatly reduce or eliminate the deleterious sideeffects that can accompany NMDA antagonists (such as convulsionpotentiating effects, as well as vacuole formation, mitochondrialdissolution, and possible death of cingulate/retrosplenialcerebrocortical neurons), without interfering with the useful propertiesof the NMDA antagonists. The protective agents described herein may alsoreduce the neurotoxic, psychotoxic, and/or hallucinatory side effectsassociated with illegal use of drugs such as phencyclidine. Mixtures ofNMDA antagonists and anti-cholinergic agents as disclosed herein can beused for beneficial purposes, such as providing safe anesthesia orameliorating neurological disease processes, while also preventingdeleterious side effects that might otherwise be caused by the NMDAantagonists.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

This invention relates to a class of protective compounds that canreduce the neurotoxic effects of NMDA antagonists. These protectivecompounds are classified as anti-cholinergic agents, since they blockthe triggering effects of acetylcholine molecules at cholinergicreceptors. Prior to this invention, it was not realized thatanti-cholinergic agents could prevent neurotoxic effects caused byagents which affect glutamate receptors, an entirely different class ofreceptors.

As used herein, the terms "neurotoxic effects" and "deleteriousneurological effects" caused by NMDA antagonists refer to any of thefollowing: (1) the formation of observable vacuoles in any type of CNScell, if comparable vacuoles of similar number, size, or type are notdetected in neurons of the same type in healthy, untreated animals; (2)the causation of damage to, dissolution of, or other significantalterations in substantial numbers of mitochondria in CNS cells; (3) thedeath of, or necrotic signs in, significant numbers of cerebrocorticalneurons if attributable to administration of an NMDA antagonist; (4) thegeneration of psychotoxic or hallucinatory effects, such as thepsychotomimetic effects of PCP, or the "emergence reaction" suffered bysome patients who are anesthetized by ketamine; or, (5) convulsionpotentiating effects.

The anti-cholinergic agents which have been tested thus far to determinewhether they can prevent neuronal vacuole formation caused by NMDAantagonists such as MK-801 and PCP include the following compounds,which are listed in decreasing order of potency, measured by theirability to prevent vacuole formation by MK-801 administered at 0.4 mg/kg(i.e., 0.4 milligrams of MK-801 per kilogram of animal body weight, asdescribed in Example 1):

1. Scopolamine provides the highest degree of protection discovered todate. It was completely effective on all animals tested to date at 0.25mg/kg.

2. Benztropine provided complete protection for all animals tested todate at 2 mg/kg.

3. Trihexyphenidyl and atropine protected all animals tested to datewhen administered at 5 mg/kg.

4. Biperiden, procyclidine, and benactyzine protected all animals at 10mg/kg.

5. Diphenhydramine, which is usually classed as an H1 histamineantagonist but which also has anti-cholinergic activity, protected allanimals at 25 mg/kg.

Additional information is contained in Table 1. The test data are basedon limited numbers of tests done to date, using the procedures describedin the Examples. As will be recognized by those skilled in the art,these data will need to be confirmed and carefully evaluated by moreextensive testing; however, the results obtained thus far are quiteclear and highly significant.

A second key finding is that the anti-cholinergic agents listed aboveand in Table 1, at doses insufficient to totally prevent vacuoleformation, substantially decreased both the number and the size of thecingulate/retrosplenial vacuoles that were caused by the MK-801. Thisresult is not reflected in the tables, which indicate only the presenceor absence of any detectable vacuoles. Clearly, a substantial reductionin the number and/or the size of vacuoles indicates that the neurotoxicproperties or activities that are manifested by vacuole formation arebeing mitigated by the anti-cholinergic agents.

Some anti-cholinergic agents (such as dicyclomine and pirenzepine) maybe of limited value for protecting against NMDA antagonistneurotoxicity, since they bind only weakly to cholinergic receptors inthe brain. It should be noted that dicyclomine and pirenzepine are oftenreferred to in the literature as "highly selective" M1 antagonists. Thiscan be confusing, since their affinity for M1 receptors is relativelyweak compared to other M1 antagonists such as scopolamine. The "highlyselective" M1 attribute derives from the fact that they have extremelyweak affinity for M2 receptors, as indicated in Table 2, which isderived from Freedman 1988 at page 136. The K_(app) values provided byFreedman et al are, in effect, dissociation values; a high K_(app) valueindicates weak binding. Therefore, the value 1/K_(app) provides anindicator of affinity. As can be seen from Table 2, although dicyclomineand pirenzpine have high M1/M2 ratios, their affinity for M1 receptorsis only a small fraction of the affinity of other compounds such asscopolamine.

In addition, preliminary experiments indicate that N-methylscopolamineis of virtually no value for use as described herein, even though it isa potent anti-cholinergic agent. This was expected, sincemethylscopolamine does not penetrate the blood-brain barrier insubstantial quantities.

The data in Tables 1 and 2 suggest that the blocking of M1 receptors isheavily involved in the neuroprotective action of anti-cholinergicagents. This follows from the data indicating that atropine offered alower degree of protection than scopolamine. However, all of theanti-cholinergics tested to date have a substantial degree of affinityfor both M1 and M2 receptors.

As mentioned previously, it is not known whether or to what extent anyanti-cholinergic agents bind to nicotinic receptors inside the CNS,because of the lack of appropriate methods for studying nicotinicreceptors within the CNS. As the state of knowledge regarding nicotinicreceptors advances, any anti-cholinergic agents which pass through theblood-brain barrier and which are reliably identified as antagonists ofCNS nicotinic receptors can be screened for protective activity againstthe neurotoxic effects of NMDA antagonists such as MK-801, using theprocedures described herein. Despite the current limited knowledgeregarding nicotinic receptors, every anti-cholinergic agent tested todate which can penetrate the blood-brain barrier has been demonstratedto be effective in reducing the neurotoxic side effects of MK-801.

One of the anti-cholinergic compounds (biperiden) has also been shown tobe effective in preventing vacuole formation by phencyclidine (PCP), asdescribed in Example 3. Since PCP and MK-801 both bind to and activatePCP receptors in the CNS, it is believed that any anti-cholinergiccompounds which provide effective protection against the side effects ofMK-801 will also protect against PCP neurotoxicity, and therefore couldbe used to counteract the permanent damage and possibly the psychoticsymptoms of PCP ("angel dust") when abused by illegal users. Theanti-cholinergic compounds discussed herein have also been tested toensure that they do not interfere with the desirable neuroprotectiveproperties of MK-801, an agent that shows great promise in blockingexcitotoxic damage involving glutamate receptors. As described inExample 4, the results are quite favorable; the anti-cholinergic agentstested to date apparently do not interfere with the beneficial effectsof MK-801.

Another set of experiments, described in Example 5, indicate that NMDAantagonists may have convulsion-potentiating effects when cholinergicreceptors are being excessively stimulated. The NMDA antagonist used(MK-801) lowered the seizure threshold and made animals more susceptibleto seizures, when the animals were treated with a cholinergic agonist(pilocarpine) at a dosage that would not have caused seizures in theabsence of the NMDA antagonist. This finding raises questions aboutwhether NMDA antagonists would also tend to induce seizures in humanswho suffer from epilepsy. However, it was also discovered that theadministration of biperiden, an anti-cholinergic, along with MK-801 andpilocarpine, protected the animals against any seizure activity, braindamage, and vacuole formation in cerebrocortical neurons. This resultindicates that the vacuole-inducing properties and the convulsionpotentiating properties of an NMDA antagonist such as MK-801 can beprevented by an anti-cholinergic agent.

A preferred embodiment of the subject invention comprises a mixture of(1) an NMDA antagonist such as MK-801, and (2) an anti-cholinergicprotective agent. The NMDA antagonist can be used for beneficialpurposes such as human or veterinary anesthesia or to protect againstexcitotoxic or neurodegenerative processes associated with variousconditions described above. The anti-cholinergic agent will prevent orminimize deleterious side effects (such as convulsions, hallucinations,or pathological changes or necrosis of cerebrocortical neurons) thatmight otherwise be caused by the NMDA antagonist. By reducing thedeleterious side effects of the NMDA antagonist without interfering withits useful activity, the anti-cholinergic agent can render the mixture amore safe and effective formulation. Included within the family ofanti-cholinergic agents useful for the purposes described herein are anytautomeric or isomeric forms or analogs which function as cholinergicantagonists (i.e., which block the activation of cholinergic receptorsby endogenous acetylcholine molecules). The anti-cholinergic activity ofany such tautomer, isomer, or analog can be tested using various methodsknown to those skilled in the art, such as competitive binding assaysusing radioactive isotopes of prototypic cholinergic agonists orantagonists, as described in, e.g., Freedman et al 1988 or Burke 1986.

Also included within the compounds of this invention arepharmaceutically acceptable salts of anti-cholinergic agents. The term"pharmaceutically-acceptable salts" embraces salts commonly used to formalkali metal salts and to form addition salts of free acids or freebases. The nature of the salt is not critical, provided that it isnon-toxic and does not substantially interfere with the anti-cholinergicactivity. Examples of acids which may be employed to formpharmaceutically acceptable acid addition salts include inorganic acidssuch as hydrochloric acid, sulphuric acid and phosphoric acid, andorganic acids such as maleic acid, succinic acid and citric acid. Othersalts include salts with alkali metals or alkaline earth metals, such assodium, potassium, calcium and magnesium. All of these salts may beprepared by conventional means.

For the purposes of this invention, it does not matter whether ananti-cholinergic agent has any other pharmaceutical effect, so long asit is capable of blocking at least one type of cholinergic receptor. Forexample, diphenhydramine is an anti-cholinergic agent even though italso has the characteristic of being an anti-histamine. Similarly, acompound is regarded as an anti-cholinergic agent if it blocks at leastone type of cholinergic receptor, regardless of whether it also blocksother types of cholinergic receptors.

The anti-cholinergic compounds tested to date are all commerciallyavailable; in addition, methods of synthesis of these compounds areknown to those skilled in the art. For example, synthesis ofprocyclidine and its salts are shown in U.S. Pat. Nos. 2,891,890 and2,826,590. Synthesis of trihexyphenidyl hydrochloride is described inU.S. Pat. No. 2,682,543. Synthesis of biperiden is described in U.S.Pat. No. 2,789,110.

Administration of anti-cholinergic compounds to humans can be by anytechnique capable of introducing the compounds into the bloodstream of ahuman patient, including oral administration or intravenous,intramuscular and subcutaneous injections. The active compound isusually administered in a pharmaceutical formulation such as in a liquidcarrier for injection, or in capsule form for ingestion, although insome acute-care situations an anti-cholinergic agent might be injectedwithout a diluting agent. Such formulations may comprise the activecompound (or a mixture of more than one anti-cholinergic compounds)together with one or more pharmaceutically acceptable carriers ordiluents. Other therapeutic agents may also be present in theformulation. Delivery of the active compound in such formulations may beby various routes including oral, nasal, topical, buccal and sublingual,or by parenteral administration such as subcutaneous, intramuscular,intravenous, or intradermal routes.

EXAMPLES EXAMPLE 1: TESTING ANTI-CHOLINERGICS AT VARYING DOSAGES

Adult female Sprague Dawley rats, approximately 4 months old, were usedin these experiments. It was previously established (Olney et al 1989)that the ED₅₀ for producing vacuoles in female Sprague Dawley rats is0.18 mg/kg of MK-801 when administered subcutaneously (sc). In thoseearlier experiments, MK-801 produced the vacuole effect in 75% of theanimals at 0.2 mg/kg sc, and in 100% at doses of 0.3 and 0.4 mg/kg sc.In the present experiment, MK-801 was administered at a dose of 0.4mg/kg sc, a dosage more than high enough to produce vacuoles in allanimals.

Ten minutes after the MK-801 was administered, the rats received anintraperitoneal (ip) injection of scopolamine, benztropine,trihexyphenidyl, atropine, biperiden, procyclidine, benactyzine, ordiphenhydramine, at a range of different doses shown in Table 1. Fouranimals were used in each treatment group, with a concurrent controlgroup (MK-801 alone) for each test compound.

It was previously established (Olney et al 1989) that the vacuole effectbecomes clearly observable within 4 hours following MK-801 treatment.Therefore, the rats were anesthetized and sacrificed by perfusionfixation of the CNS at 4 hours following MK-801 treatment, using amixture of glutaraldehyde and paraformaldehyde injected into the leftcardiac ventricle. Their brains were processed by methods permittinghistopathological evaluation by both light and electron microscopy, asdescribed in Olney 1971. The tissue samples were evaluated usingnumerical codes, by an experienced histopathologist who had no knowledgeof the treatment conditions for any given tissue sample.

In all control rats (32 of 32), a severe vacuole reaction incingulate/retrosplenial cerebrocortical neurons was observed. Asindicated in Table 1, all of the anti-cholinergic agents preventedvacuole formation in a dose-related manner. All except diphenhydramineprovided total protection at doses above 5 mg/kg, and atropine,trihexyphenidyl, benztropine, and scopolamine gave complete protectionat doses above 2, 2, 1, and 0.1 mg/kg, respectively. When vacuoles werepresent in rats treated with doses below the thresholds for completeprotection, the number and size of the vacuoles usually appeared smallerthan in the control rats. Thus, the degree of protection conferred isconservatively stated when described in terms of the number of brainswith vacuoles.

EXAMPLE 2: TESTING MK-801 AT VARYING DOSAGES

The lowest dose of biperiden (10 mg/kg) that provided completeprotection against MK-801 vacuole formation in Example 1 wasadministered to rats that received MK-801 on an increasing dose schedule(1, 2.5 and 5 mg/kg). It was found that the same low dose of biperiden(10 mg/kg) that had protected against 0.4 mg/kg MK-801 in Example #1also protected completely against doses of MK-801 up to and including 5mg/kg. In all experiments, the biperiden was well-tolerated by the rats.The Physicians Desk Reference indicates that the LD₅₀ for biperiden inadult rats is 270 mg/kg, so the margin of safety between effective andtoxic doses is quite favorable.

EXAMPLE 3: PCP TESTING

In order to determine whether anti-cholinergic agents protect againstthe vacuole-forming neurotoxicity of NMDA antagonists other than MK-801,female adult Sprague Dawley rats were treated with PCP (10 mg/kg sc).Ten minutes later, they were treated with biperiden at doses of 10, 5,and 1 mg/kg ip. A dose-related protective effect was demonstrated. PCPcaused a severe vacuole reaction in all control rats (4/4), and in 0/4,0/4, and 1/4 rats treated with biperiden at 10, 5, and 1 mg/kgrespectively.

EXAMPLE 4: NON-INTERFERENCE WITH DESIRED MK-801 EFFECTS, USING NMDA ATEXCITOTOXIC LEVELS

To determine whether anti-cholinergics interfere with the ability ofMK-801 to protect neurons against the excitotoxic action of NMDA, an exvivo chick embryo retina assay was used. This preparation has been founduseful for studying the excitotoxic effects of NMDA on CNS neurons andfor evaluating the ability of antagonists to protect against NMDAexcitotoxicity (Olney 1989). In these experiments, 18 segments of chickretina were incubated, each in a separate well, in medium containingNMDA at a concentration of 80 micromolar (uM), which previously had beendetermined to produce a fully developed excitotoxic lesion in 30minutes. MK-801 was added to 12 wells at 0.2 uM, a thresholdconcentration known to consistently block the neurotoxic action of NMDA;in 6 of those wells, both MK-801 (0.2 uM) and biperiden (50 uM) wereadded.

In the six wells containing NMDA but no MK-801, a full retinal lesionwas present within 30 minutes. In all twelve wells that contained bothNMDA and MK-801, the MK-801 blocked the neurotoxic action of the NMDA,either in the presence of biperiden (6/6) or in absence of biperiden(6/6). Therefore, even when biperiden was present in .CNS tissue at aconcentration 250 times higher than the concentration of MK-801, it didnot prevent the MK-801 from exerting its neuroprotective action againstthe excitotoxic processes mediated by NMDA triggering of NMDA receptors.

In Example 2, it required a dose of only 10 mg/kg biperiden tocompletely block the neurotoxic effects of 5 mg/kg MK-801 oncingulate/retrosplenial cortical neurons. Thus, when BPN and MK-801 arepresent in a 2:1 ratio, biperiden protects against MK-801's neurotoxicside effects, but when present in a 250:1 ratio, biperiden does notinterfere with MK-801's ability to potect against NMDA receptor-mediatedneurodegenerative processes.

EXAMPLE 5: DEMONSTRATION OF THE CONVULSION POTENTIATING EFFECTS OFMK-801, AND PROTECTION USING BIPERIDEN

Pilocarpine is a cholinergic agonist which causes seizures andseizure-related brain damage. It is used in research pertaining toepilepsy, since it is believed that the receptor mediated processesinvolved in pilocarpine-induced seizures are similar in some respects tothe mechanisms of epileptic seizures. Although the pilocarpine dosagethat induces seizures and seizure-related brain damage varies betweenindividual rats, it is usually in the range of about 380-400 mg/kg sc.

The Applicant previously established that MK-801 can reduce or eliminateseizures and seizure-related brain damage caused by various methods(Clifford et al 1989). However, when the inventor tried to use MK-801 toreduce seizures induced by pilocarpine, he discovered that thecombination of MK-801 and pilocarpine resulted in a severe seizure-braindamage syndrome at relatively low doses (50 mg/kg sc pilocarpine,injected 10 minutes after 1 mg/kg sc MK-801). At those doses, neitheragent by itself can cause seizures or seizure-related brain damage. TheMK-801 therefore potentiates the effects of pilocarpine, i.e., it lowersthe seizure threshold and makes the animal more susceptible to aseizure. These observations--that MK-801 protects against some types ofseizure-brain damage syndrome but potentiates other suchsyndromes--suggest that MK-801 might have unpredictable and in somecases adverse effects in humans who are subject to epileptic seizures,and in humans who are being treated with various neuropharmacologicaldrugs.

Adult rats were also treated with both MK-801 (1 mg/kg sc) and biperiden(10 mg/kg ip); ten minutes later, they were injected with pilocarpine(50 mg/kg sc). The additional presence of the biperiden prevented allneurotoxic manifestations, including seizure activity, the widespreadbrain damage that would otherwise have been caused by the pilocarpineand MK-801, and the formation of vacuoles in cingulate/retrosplenialneurons.

The observation that MK-801 potentiates the convulsant effects ofpilocarpine adds further evidence that a linking mechanism connects theexcitotoxic processes that involve cholinergic receptors and NMDAreceptors. The discovery that a cholinergic antagonist can block theconvulsion potentiating effects of MK-801 further confirms that link,and indicates that by combining an anti-cholinergic agent with an NMDAantagonist such as MK-801, it is possible to utilize the anti-convulsiveproperties of both agents while eliminating the convulsion potentiatingeffects of the NMDA antagonist.

Thus, there has been disclosed a class of pharmacological agents whichcan function safely and effectively in accomplishing beneficial resultswhich were not previously available. This invention therefore satisfiesall of the objectives set forth herein. Although this invention has beendescribed with respect to specific embodiments, the details of theseembodiments are not to be construed as limitations. Various equivalentsand modifications may be made without departing from the spirit andscope of this invention, which is limited only by the claims whichfollow.

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I claim:
 1. A pharmacological composition comprising a mixture of anNMDA antagonist and an anti-cholinergic agent, both of which canpenetrate blood-brain barriers, wherein the NMDA antagonist is presentin a therapeutically effective quantity sufficient to reduce excitotoxicdamage in the brain if administered to a mammal, and wherein the NMDAantagonist can cause neurotoxic side effects in the brain ifadministered without an accompanying anti-cholinergic agent, and whereinthe anti-cholinergic agent is present in a second quantity that canreduce the neurotoxic side effects which would be caused by the NMDAantagonist if administered without the accompanying anti-cholinergicagent.
 2. A pharmacological composition of claim 1, wherein the NMDAantagonist is selected from the group consisting of(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate(common name MK-801), phencyclidine, ketamine, and tiletamine.
 3. Apharmacological composition of claim 1, wherein the anti-cholinergicagent exerts a pharmaceutically antagonistic effect on cholinergicreceptors of the M1 muscarinic type on the surfaces of neurons in thecentral nervous system.
 4. A pharmacological composition of claim 2,wherein the anti-cholinergic agent exerts a pharmaceuticallyantagonistic effect on cholinergic receptors of the M1 muscarinic typeon the surfaces of neurons in the central nervous system.
 5. Apharmacological composition of claim 1, wherein the anti-cholinergicagent is selected from the group consisting of scopolamine, atropine,benztropine, benactyzine, biperiden, procyclidine, trihexyphenidyl, anddiphenhydramine, and pharmaceutically acceptable salts thereof.
 6. Apharmacological composition of claim 2, wherein the anti-cholinergicagent is selected from the group consisting of scopolamine, atropine,benztropine, benactyzine, biperiden, procyclidine, trihexyphenidyl, anddiphenhydramine, and pharmaceutically acceptable salts thereof.
 7. Apharmacological composition of claim 3, wherein the anti-cholinergicagent is selected from the group consisting of scopolamine, atropine,benztropine, benactyzine, biperiden, procyclidine, trihexyphenidyl, anddiphenhydramine, and pharmaceutically acceptable salts thereof.